2000 Progress Report: Metabolic Engineering of Solvent Tolerance in Anaerobic BacteriaEPA Grant Number: R828562
Title: Metabolic Engineering of Solvent Tolerance in Anaerobic Bacteria
Investigators: Papoutsakis, E. T. , Welker, N. E.
Institution: Northwestern University
EPA Project Officer: Richards, April
Project Period: June 1, 2000 through May 31, 2003
Project Period Covered by this Report: June 1, 2000 through May 31, 2001
Project Amount: $180,000
RFA: Technology for a Sustainable Environment (1999) RFA Text | Recipients Lists
Research Category: Sustainability , Pollution Prevention/Sustainable Development
Objective:Understanding solvent (and other toxic chemical) tolerance of microorganisms is crucial for the production of chemicals, bioremediation, and whole-cell biocatalysis. Also, it is very important basic knowledge. Past efforts to produce tolerant strains have relied on selection under applied pressure and chemical mutagenesis, with some good results, but not consistently so. We will examine Metabolic Engineering (ME) and genomic approaches to determine if they can be used to construct more tolerant strains for bioprocessing. The accepted dogma is that toxicity is due to the chaotropic effects of solvents on the cell membrane. Impaired membrane fluidity and function inhibit cell metabolism, and result in cell death. We have found that in Clostridium acetobutylicum, several well-defined genetic modifications not related to membrane function impart solvent tolerance (by 40-70 percent) without strain selection. This suggests that we need to reexamine the accepted dogma. The objective of this research is to identify genes that contribute to solvent tolerance and to use genetic modifications (involving these genes) to generate solvent tolerant strains. In view of the large number of possible genes that may be involved in determining solvent tolerance, we will use DNA microarrays for transcriptional analysis.
Our progress is described below.
1. Overexpression of orfa, a Putative Repressor of the GroESL and DnaKJ Operons
The major heat shock proteins DnaKJ and GroESL are essential to normal cellular function. They bind proteins in non-native states and assist in their proper folding. The role and regulation of the DnaKJ and GroESL protein families in C. acetobutylicum is not well understood. orfA is a putative repressor of the GroESL and DnaKJ operons, as identified by homology to hrcA in Bacillus subtilis. hrcA has been shown to operate as a negative repressor through interaction with a CIRCE (Controlling Inverted Repeat of Chaperone Expression) element located upstream of both the DnaKJ and GroESL operons. The orfA gene was cloned from the DnaKJ operon using PCR primers that amplified the structural sequence, but not the regulatory sequences. The orfA gene was ligated to an Escherichia coli/C. acetobutylicum shuttle vector such that orfA is under the control of the clostridial thiolase (thl) strong promoter. The resulting plasmid (porfA1) was transformed into C. acetobutylicum ATCC 824. Northern Blot analysis of the orfA overexpression strain 824 (porfA1) was compared to that of the wild type 824 strain to verify increased transcription of orfA from porfA1. A probe designed to detect the orfA gene resulted in a strong 1.1kb band present in the 824 (porfA1) samples, but not in the wild type 824 samples. The same orfA probe also resulted in a 3.8kb band present in the wild type samples, that was not present in the 824 (porfA1) samples. The 3.8kb band corresponds to the expected 3.8kb transcript from the DnaKJ operon. The fact that this transcript is absent in the orfA overexpressing strain is expected if the orfA protein is in fact acting as a repressor of the DnaKJ operon. A second probe to the dnaK/dnaJ genes confirmed this finding. A third probe to the GroESL operon resulted in a 2.2kb band corresponding to the expected transcript size. The 2.2kb band was an order of magnitude more intense on the wild type blot relative to the orfA overexpressing strain. This is the first direct evidence that orfA acts as a repressor of the DnaKJ and GroESL operons in C. acetobutylicum.
2. Overexpression of DnaKJ and GroESL Operon Genes
We cloned a fragment of the DnaKJ operon containing all three functional genes (grpE, dnaK, and dnaJ). Similarly, the GroESL operon genes (groES and groEL) were amplified without its natural promoter or the CIRCE element. The amplified fragments have been ligated to two E. coli/C. acetobutylicum shuttle vectors under control of the ptb (phosphotransbutyrylase) or the thl (thiolase) promoters. Initial efforts to identify an E. coli clone in which the GroESL and DnaKJ genes were successfully inserted into the vector did not result in a positive clone. It has been shown that overproduction of HSPs is toxic in a number of hosts (including E. coli and B. subtilis). We hypothesized that transformation and growth of E. coli at a lower temperature (25oC) may decrease the initial level of stress created by increased HSP production. This hypothesis proved to be correct in so far as it resulted in the identification of positive clones: positive clones grew up to 1,000 times better at 25?C versus cells grown at 37?C, as determined by CFU/mL counts. Before C. acetobutylicum ATCC 824 can be transformed with these four constructs, it is necessary to methylate the constructs by first passing them through the methylating strain E. coli ER2275 (pAN1). Plasmid pAN1 contains the f3T I methyltransferase gene that methylates Cac824I restriction sites. Similar temperature-related difficulties have been encountered with this transformation. One of the four constructs (pGROE1 ? thl promoter) has been methylated and inserted into C. acetobutylicum. A new strategy is now undertaken to make methylation of these plasmids possible. Rather than transforming the desired plasmid into E. coli in which pAN1 is already established, E. coli carrying the desired plasmid will be made electrocompetent and then transformed with pAN1.
3. Expression Analysis of the Butanol-Stress Response Using DNA Arrays
A DNA-microarray protocol has been developed for expression analysis of C. acetobutylicum. We constructed DNA arrays containing 96 to 500 genes, all PCR amplified from C. acetobutylicum. Genes have been selected from the following classes/pathways: glycolysis and sugar metabolism, solvent and acid production, the heat-shock response, antibiotic resistance, sporulation, and energy generation. The arrays also contain several positive and negative controls. The expression profile of both butanol- and heat-stressed cells was determined. Cells were exposed to butanol (1percent) and heat (42?C) for 10 minutes. The data show that transcription levels change after only 10 minutes of stress. Exposure to butanol alters the expression of genes that have been implicated in the stress response (chaperonins) and of other genes that usually are not implicated in stress pathways. Some genes involved in butanol synthesis were upregulated, while other solvent-production related genes had relative low transcript levels.
The following future activites will be performed:
Overexpression of DnaKJ and GroESL. Plasmid constructs for overexpression of DnaKJ and GroESL genes will be methylated and transformed into C. acetobutylicum ATCC 824. A new strategy, outlined above, will be used to successfully methylate and transform Clostridia with these constructs. These strains will be characterized by fermentation and flux analysis as well as by Northern and Western analysis. DNA microarrays also will be utilized to examine the effect of HSP overexpression on global gene expression.
Identification and Study of Other Genes That Might Be Involved in Solvent Tolerance. We will manufacture larger DNA arrays (to examine the expression profile of 1,100 genes; this will cover about 25 percent of the C. acetobutylicum genome) to examine the effects of solvent challenges on the expression of a large number of genes. Genes that show large differences in differential expression will be cloned and characterized, and their overexpression will be examined for the ability to confer increased solvent tolerance using an approach identical to the one described above for the HSPs.